Kavalactones and Benzoic Acid Derivatives from Leaves of Piper fuligineum Kunth (Piperaceae)

Abstract

The known kavalactones (E)-4-methoxy-6-styryl-2H-pyran-2-one, 4-methoxy-6-(3-phenyloxiran-2-yl)-2H-pyran-2-one, 6-(1,2-dihydroxy-2-phenylethyl)-4-methoxy-2H-pyran-2-one, the three benzoic acid derivatives methyl-4-methoxy-3-(3'-methyl-2'-butenyl)benzoate and methyl 2,2-dimethyl-4-oxochroman-6-carboxylate, and a new methyl 4-methoxy-3-(3-methylbut-2-enoyl)benzoate were isolated from the ethanolic extract of Piper fuligineum. The structures of these compounds were determined by using a combination of spectroscopic methods, including 1D- and 2D-nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. This is the first report of the chemical study of P. fuligineum, and the methyl 4-methoxy-3-(3-methylbut-2-enoyl)benzoate is described as a new natural product.

Keywords:
Piper fuligineum; Piperaceae; kavalactones; benzoic acid derivatives

Introduction

Studies of Piper species have revealed the presence of several classes of secondary metabolites,¹ including alkaloids, amides,^2-6 chromenes,^7,8 neolignans,⁹ lignans, terpenes,^10,11 benzoic acid derivatives^12,13 and kavalactones.¹⁴ Many of these compounds have shown important biological activities, including antifungal,⁴ anti-inflammatory,¹⁵ antiparasitic,¹³ antioxidant,¹⁶ anticoagulant¹⁷ and trypanocidal properties.^18-20 The rhizome of Piper methysticum, known as “kava-kava” in Hawaii and the South Pacific Islands, is used to prepare a beverage to treat anxiety.^21,22 The major compounds isolated from this species are the kavalactones kavain and methysticin. Although it has been banned in the UK and several other countries due to its potential hepatotoxicity, the mechanism for this hepatotoxicity is still unclear.²³ To date, the occurrence of this class of compounds is limited to a few Piper species, including P. rusbyi,²⁴P. sanctum,²⁵P. cubeba²⁶ and P. dilatatum.¹⁴

Piper fuligineum Kunth is a shrub endemic to Brazil that grows mainly in Cerrado, Caatinga and Atlantic Forest.^27-30 This species was previously found to contain the kavalactone demethoxy-yagonin ((E)-4-methoxy-6-styryl-2H-pyran-2-one (1)), which showed inhibitory activity towards hepatitis C virus replication.³¹ In addition to 1, we also report the isolation of two kavalactones from P. fuligineum leaves, 4-methoxy-6-(3-phenyloxiran-2-yl)-2H-pyran-2-one (2) and 6-(1,2-dihydroxy-2-phenylethyl)-4-methoxy-2H-pyran-2-one (3), as well as three benzoic acid derivatives, methyl-4-methoxy-3-(3'-methyl-2'-butenyl)benzoate (4), methyl 2,2-dimethyl-4-oxochroman-6-carboxylate (5), and methyl 4-methoxy-3-(3-methylbut-2-enoyl)benzoate (6). Compounds 1-5 were previously isolated from Piper species, but compound 6 is, to the best of our knowledge, a new natural product.

Experimental

General

One-dimensional (¹H, ¹³C) and two-dimensional (gHMBC (gradient heteronuclear multiple bond correlation) and gHMQC (gradient heteronuclear multiple quantum correlation)) spectra were recorded on a Varian Inova-500 (11.7 T) spectrometer at 500 MHz (¹H) and 125 MHz (¹³C) using CDCl₃ as a solvent and TMS as a reference. HR-ESI-MS (high-resolution electrospray ionization mass spectrometry) was measured using a Bruker Daltonics model ultrOTOF_Q ESI-TOF (time of flight) instrument. Separations by column chromatography (CC) were carried out using silica gel (230-400 mesh; Merck). All solvents were distilled prior to use. High-performance liquid chromatography (HPLC) separations were performed on a Varian PrepStar model SD-1 LC/UV/VIS chromatograph equipped with a Phenomenex C-18 reversed phase column (250 × 21.2 mm).

Plant material

The leaves of P. fuligineum Kunth were collected at the Chácara Flora (Araraquara-SP, Brazil) in October of 2006 and identified by Dr Inês Cordeiro. A voucher specimen (Kato-0720) has been deposited at the Herbarium of the Instituto de Botânica of Universidade de São Paulo (São Paulo-SP, Brazil).

Isolation of compounds

Dried leaves (420 g) of P. fuligineum were milled, extracted with EtOH, and it was concentrated under vacuum to yield 54.4 g of the extract. The extract was resuspended in MeOH:H₂O (4:1) and partitioned with hexane, CHCl₃ and EtOAc sequentially. The portion soluble in CHCl₃ (13.0 g) was subjected to CC over silica gel and eluted with a gradient of hexanes-EtOAc to yield fractions 1-23. Fraction 5 (2.15 g) was submitted to flash CC over silica gel eluted with a gradient of hexanes-EtOAc yielding sub-fractions 5-1 to 5-28. Sub-fraction 5-1 (320.3 mg) was submitted to preparative HPLC eluted with MeOH:H₂O (1:1) to afford the compounds 6 (4.6 mg), 4 (7.4 mg) and 5 (5.8 mg). Fraction 8 (2.0 g) was subjected to CC over silica gel and eluted with a gradient of n-hexanes-EtOAc providing fractions 8-1 to 8-16. Sub-fraction 8-4 afforded1(58.8 mg). Fraction 16 (1.3 g) was subjected to preparative HPLC and eluted with isocratic MeOH:H₂O (65:35) to afford 3 (2.3 mg).

Methyl 4-methoxy-3-(3-methylbut-2-enoyl)benzoate (6)

Amorphous white powder, UV (MeOH) λ_max / nm 253, 305; ¹H and ¹³C nuclear magnetic resonance (NMR), see Table 1. HRMS/ESI-TOF m/z (rel. int.): 249.1129 [M + H]⁺ (80) (calcd. for C₁₄H₁₇O₄, 249.1126); 271.0949 [M + Na]⁺ (98) (calcd. for C₁₄H₁₆O₄Na, 271.0946).

Table 1
¹H and ¹³C NMR spectroscopic data for compound 6 (500 MHz, CDCl₃, δ in ppm, J in Hz), isolated from P. fuligineum

Position	δC	δH (mult; J in Hz)	gHMBC
1	130.9	-
2	131.7	8.14 (d; 2.5; 1H)	C1", C1'
3	122.6	-
4	161.0	-
5	111.2	6.91 (d; 8.5; 1H)
6	133.9	8.03 (dd; 8.5 and 2.5; 1H)	C1", C4
7	55.9	3.87 (s; 3H)	C4
1'	191.9	-
2'	124.8	6.49 (m; 1H)	C1', C5'
3'	156.5	-
4'	21.3	2.16 (d; 1.0; 3H)	C1', C2', C3', C5'
5'	28.0	1.90 (d; 1.0; 3H)	C2', C3'
1"	166.3	-
2"	52.0	3.82 (s; 3H)	C1"

Results and Discussion

The EtOH extract from the leaves of P. fuligineum was suspended in MeOH:H₂O (4:1) and sequentially partitioned against the hexanes, CHCl₃ and EtOAc. ¹H NMR analysis of the concentrated fractions revealed signals of aromatic compounds (d 6-8) in the CHCl₃ fraction and more signals than in hexanes or EtOAc. Thus, the CHCl₃ soluble fraction was submitted to a chromatographic purification procedure, which yielded three kavalactones (1-3) and three benzoic acid derivatives (4-6).

The known compounds were identified as (E)-4-methoxy-6-styryl-2H-pyran-2-one (1),^25,26,31 4-methoxy-6-(3-phenyloxiran-2-yl)-2H-pyran-2-one (2), 6-(1,2-dihydroxy-2-phenylethyl)-4-methoxy-2H-pyran-2-one (3),²⁴ methyl-4-methoxy-3-(3'-methyl-2'-butenyl)benzoate (4)^32,33 and methyl 2,2-dimethyl-4-oxochroman-6-carboxylate (5) by NMR and/or by HRMS (Supplementary Information) as well as by comparison with the literature data.^{13,14,24,34,35}

The molecular formula of 6 was established as C₁₄H₁₆O₄ by HRMS ([M + H]⁺ observed m/z 249.1129, calcd. 249.1126; [M + Na]⁺ observed m/z 271.0949, calcd. 271.0946), which matched the overall ¹H and ¹³C NMR analysis. The ¹H NMR spectrum (Table 1) exhibited resonances indicative of one meta-coupled aromatic hydrogen at d 8.14 (H-2, d, J 2.5 Hz, 1H), one ortho-coupled at d 6.91 (H-5, d, J 8.5 Hz, 1H), and one ortho-meta-coupled at d 8.03 (H-6, dd, J 8.5 and 2.5 Hz, 1H). Doublets at d 1.90 (H-5', d, J 1.0 Hz, 3H) and 2.16 (H-4', d, J 1.0 Hz, 3H) and a multiplet at d 6.49 (H-2', m, 1H) were assigned to the oxidized prenyl group. The singlets observed at d 3.82 (H-2”, s, 3H) and 3.87 (H-7, s, 3H) corresponded to the methyl ester and aromatic methoxyl groups, respectively. The oxidized prenyl group was confirmed by the gHMBC and gHMQC cross-peaks of d 1.90 (H-5') and 2.16 (H-4') to C-2' (d 124.8). In addition, cross-peaks of both H-6 (d 8.03) and H-2 (d 8.14) to C-1” (d 166.3) were observed, thus confirming the ester substituent at the C-1 position of the aromatic ring. The cross-peak of H-7 (d 3.87) to C-4 (d 161.0) corroborated the placement of the methoxy group. The ¹³C NMR data corroborated the substituents of aromatic ring, and all signals were assigned accordingly based on the gHMBC data (Table 1). Thus, compound 6 was established as methyl 4-methoxy-3-(3-methylbut-2-enoyl)benzoate, a methylated derivative of methyl taboganate.³⁶

The biogenesis involved in the formation of kavalactones and benzoic acid derivatives in P. fuligineum suggests that shikimic acid is the key building block in their formation (Figure 1). Additionally, the oxidation level of the benzoic acid derivatives in P. fuligineum indicates that such oxidations are important processes for achieving the chemical diversity observed for the kavalactones and benzoic acid derivatives, which includes a chromanone. The biosynthetic pathways in Piper fuligineum suggest that this species is an important target whose capacity to produce kavalactones similar to other Piper species should be explored and that can be used to discover new pharmacological applications.

Figure 1
Compounds isolated from P. fuligineum (1-6) and their biosynthetic relationships. IPP: isopentenyl diphosphate; DMAPP: 3,3-dimethylallyl diphosphate.

Conclusions

In this work, we describe the identification and structural elucidation of six compounds from the leaves of P. fuligineum, including one new prenylated benzoic acid derivative in addition to kavalactones, which represent an important class of compounds, but occur in only a few specific plant species. The characterization of these compounds expands our knowledge on the chemical diversity of Piper species.

Supplementary Information

Supplementary information is available free of charge at http://jbcs.org.br as PDF file.

https://minio.scielo.br/documentstore/1678-4790/rwkTJsqks58nPMy3K4H7VNN/f854d5ad2cf42dec43c164f9845eaa50c0eb0255.pdf

Acknowledgments

This work was supported by grants from the State of São Paulo Research Foundation (FAPESP): Research, Innovation and Dissemination Centers (CEPID, CIBFar-2013/07600-3) as well as 05/51850-9 and 14/50316-7. M. F. and M. J. K. are grateful to CNPq for research fellowships. B. F. M., L. G. F. and F. C. thank FAPESP for the provision of scholarship and fellowship, respectively (2011/16752-6, 2013/15306-8 and 2007/56140-4).

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Publication Dates

• Publication in this collection
  June 2018

History

• Received
  30 June 2017
• Accepted
  6 Dec 2017